p38MAPK guards the integrity of endosomal compartments through regulating necrotic death

Pathogens trigger activation of sensors of the innate immune system that initiate molecular signaling enabling appropriate host defense programs. Although recognition of pathogen-specific moieties or PAMPs by specialized receptors of the immune system is well defined for a great number of pathogens, the mechanisms of sensing of pathogen-induced functional perturbations to the host cell remain poorly understood. Here we show that the disruption of endosomal compartments in macrophages by a bacterium or fully synthetic nanoparticles activates stress-response p38MAPK kinase, which triggers execution of cell death of a necrotic type. p38MAPK-mediated necrosis occurs in cells with a compound homozygous deletion of pyroptosis-inducing caspases-1 and -11, apoptotic caspase-8, and necroptosis-inducing receptor-interacting protein kinase-3 (RIPK3), indicating that all of these principal cell death mediators are dispensable for p38MAPK-induced necrosis in response to endosome rupture. p38MAPK-mediated necrosis is suppressed by the receptor-interacting protein kinase 1, RIPK1, and degradation of RIPK1 sensitizes macrophages to necrotic death. Since pathogen-induced cell death of necrotic types is implicated in host defense against infection, our results indicate that functional perturbations in host cells are sensed as a component of the innate immune system.

Legends to Supplemental Movies S1 and S2.

Movie S1
Treatment of BMDMs from MLKL-deficient mice with TM alone triggers no necrotic cell death, as determined by the addition to cell media PI (red). Time-lapsed images of treated cells were obtained with IncuCyte imaging system.

Movie S2
Treatment of BMDMs from MLKL-deficient mice with TM and NPs triggers necrotic-type cell death, as determined by the addition to cell media PI (red). Time-lapsed images of treated cells were obtained with IncuCyte imaging system. Tables S1 and S2.   Table S1.

Legends to Supplemental
The list of 500 genes that most strongly contributed to the divergence on a principal component PC-1 axis between transcriptomes of macrophages treated with tunicamycin alone compared to macrophages treated with a combination of tunicamycin and NP. Please see Methods section for more information on data processing pipeline. Table S2.
The list of biological processes over-represented in macrophages treated with a combination of tunicamycin and NP compared to macrophages treated with tunicamycin alone based on Gene ontology (GO) biological process over-representation analysis. Please see Methods section for more information on data processing pipeline.    Macrophage treatment with bacteria activates broader range of innate immune pathways than nanoparticles. The heatmap of the GSEA z-scores of the REACTOME gene sets that are enriched after BMDM treatment with NP, L.mono, or ∆HLY, compared to buffer-treated control.
The z-scores in the heatmap were clustered without supervision. The light blue color represents an imputed value of z-scores equal zero and P-values >0.05, compared to Mock buffer treated controls. BMDM cells from C57BL6/J or 129S1 mice were treated with TM and NP and cell death was assayed by LDH activity in the media, N=3. ** P=0.0025; *** P=0.0004.  variable into the raw global transcriptomics data files for each sample allows for isolation of top genes contributing to the divergence of transcriptional phenotypes between TM+NP and TMtreated cells, and leads to defined sample distribution between these two treatment conditions along PCA Dimention-1 axis. The top 500 genes contributing to separation of samples on PC-1 axis are shown in Table S1. The 22 genes most contributing to sample separation along dimention 1 axis are shown with blue arrows. N=4 for each experimental condition.    Fig. 1C in a cropped form. The areas of the membranes that were cropped and presented in Fig. 1C for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 1C. Re-probing sequence for the same membrane is indicated with blue arrow.

Caspase1 GAPDH
Caspase 8 GAPDH Ripk3 GAPDH Caspase 11 GAPDH  Fig. 2A and Fig. 2C in a cropped form. The areas of the membranes that were cropped and presented in Fig. 2A and Fig. 2C for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 2A and Fig. 2C. Re-probing sequence for the same membrane is indicated with blue arrow.

GAPDH Bak GAPDH
WT Caspase 3 GAPDH The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 2C and Fig. 2D. Re-probing sequence for the same membrane is indicated with blue arrow.

Caspase 3 GAPDH WT Caspase 7 Actin 4KO Caspase 7 Actin
Caspase7 signal from previous blotting. Fig. 2G in a cropped form. The areas of the membranes that were cropped and presented in Fig. 2G for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 2G. Re-probing sequence for the same membrane is indicated with blue arrow. Fig. 4A in a cropped form. The areas of the membranes that were cropped and presented in Fig. 4A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 4A. Re-probing sequence for the same membrane is indicated with blue arrow. Fig. 4A in a cropped form. The areas of the membranes that were cropped and presented in Fig. 4A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 4A. Re-probing sequence for the same membrane is indicated with blue arrow.

WT MK2 GAPDH
WT phospho-MK2 GAPDH The areas of the membranes that were cropped and presented in Fig. 4A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 4A. Re-probing sequence for the same membrane is indicated with blue arrow.

4KO p38 GAPDH
4KO Phospho-p38 GAPDH  Fig. 4B in a cropped form. The areas of the membranes that were cropped and presented in Fig. 4B for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 4B. Re-probing sequence for the same membrane is indicated with blue arrow.

WT MK2 p38 GAPDH
WT Phospho-p38 GAPDH  Fig. 4B in a cropped form. The areas of the membranes that were cropped and presented in Fig. 4B for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 4B. Re-probing sequence for the same membrane is indicated with blue arrow.
WT p-MK2 GAPDH Fig. S22. The full-length images of the original Western blot membranes demonstrating expression of indicated proteins, shown in Fig. 4B in a cropped form. The areas of the membranes that were cropped and presented in Fig. 4B for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 4B. Re-probing sequence for the same membrane is indicated with blue arrow.
4KO phospho-p38 GAPDH 4KO p38 MK2 and GAPDH MK2 GAPDH  Fig. 5A in a cropped form. The areas of the membranes that were cropped and presented in Fig. 5A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 5A. Re-probing sequence for the same membrane is indicated with blue arrow.

P-MK2 GAPDH p38
P-P38 GAPDH MK2 Fig. 5C in a cropped form. The areas of the membranes that were cropped and presented in Fig. 5C for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 5C. Re-probing sequence for the same membrane is indicated with blue arrow. Fig. 5E in a cropped form. The areas of the membranes that were cropped and presented in Fig. 5E for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 5E. Re-probing sequence for the same membrane is indicated with blue arrow.

WT phospho-p38 GAPDH
WT phospho-MK2 GAPDH GAPDH P-p38 GAPDH Fig. 5E in a cropped form. The areas of the membranes that were cropped and presented in Fig. 5E for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 5E. Re-probing sequence for the same membrane is indicated with blue arrow.

WT p38 MK2 GAPDH
4KO phospho-MK2 GAPDH Fig. 5E in a cropped form. The areas of the membranes that were cropped and presented in Fig. 5E for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 5E. Re-probing sequence for the same membrane is indicated with blue arrow.

4KO p38 MK2 GAPDH
4KO phospho-p38 GAPDH Fig. 6B in a cropped form. The areas of the membranes that were cropped and presented in Fig. 6B for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 6B. Re-probing sequence for the same membrane is indicated with blue arrow. Fig. 6E in a cropped form. The areas of the membranes that were cropped and presented in Fig. 6E for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 6E. Re-probing sequence for the same membrane is indicated with blue arrow.

Fig. S29. The full-length images of the original Western blot membranes demonstrating expression of indicated proteins, shown in
WT p-p38 Actin Fig. 6I and Fig. 6J in a cropped form. The areas of the membranes that were cropped and presented in Fig. 6I and Fig. 6J for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Fig. 6I and Fig. 6J. Re-probing sequence for the same membrane is indicated with blue arrow. Fig. S8A in a cropped form. The areas of the membranes that were cropped and presented in Supplemental Fig. S8A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8A. Reprobing sequence for the same membrane is indicated with blue arrow. The blotting order for p-AKT membrane was P-AKT, GAPDH, and then MK2 for Fig. 4A.

WT AKT GAPDH
WT p-AKT GAPDH Fig. S8A in a cropped form. The areas of the membranes that were cropped and presented in Supplemental Fig. S8A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8A. Reprobing sequence for the same membrane is indicated with blue arrow. Fig. S8A in a cropped form. The areas of the membranes that were cropped and presented in Supplemental Fig. S8A for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8A. Re-probing sequence for the same membrane is indicated with blue arrow. Fig. S8A and Fig. S8C in a cropped form. The areas of the membranes that were cropped and presented in Supplemental Fig. S8A and Fig.  8C for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8A and Fig. 8C. Re-probing sequence for the same membrane is indicated with blue arrow.

WT TAK1 GAPDH
WT p-ASK1 p-cJUN GAPDH  Fig. S8B for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8B. Reprobing sequence for the same membrane is indicated with blue arrow. The membrane for JNK was also used to analyze expression of ATF2 (Fig. 4A). The probing order was JNK, ATF2, GAPDH. The membrane for p-JNK was also used to analyze expression of p-AKT (S35); the order of processing was p-JNK, p-AKT, GAPDH.     Fig. S8D for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8D. Reprobing sequence for the same membrane is indicated with blue arrow. The membrane for 4KO p-ASK1 was also used for analyzing MK2 in 4KO cells (Fig4A). The probing order was MK2, pASK1, GAPDH.

4KO
4KO p-ASK1 GAPDH Fig. S42. The full-length images of the original Western blot membranes demonstrating expression of indicated proteins, shown in Supplemental Fig. S8D in a cropped form. The areas of the membranes that were cropped and presented in Supplemental Fig. S8D for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8D. Reprobing sequence for the same membrane is indicated with blue arrow. The membrane for TAK1 was used to analyze expression of p-p38 first (Fig4A). The membrane for p-TAK1 was used to analyze expression of p38 (Fig4A). The strong low molecular weight bands represent signal post p-p38 and p38 staining, respectively.

4KO TAK1 4KO p-TAK1
4KO TRAF2 GAPDH  Fig. S8D for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8D. Reprobing sequence for the same membrane is indicated with blue arrow.
4KO p-TRAF2 GAPDH Fig. S44. The full-length images of the original Western blot membranes demonstrating expression of indicated proteins, shown in Supplemental Fig. S8D in a cropped form. The areas of the membranes that were cropped and presented in Supplemental Fig. S8D for each protein are depicted with red rectangles. The image of the molecular weight ladder loaded on the same gel was taken for each original membrane and was juxtaposed with the image of the same membrane after the Western blot processing. The border of the molecular weight ladder image is depicted by the black rectangle. The molecular weights corresponding to specific bands in the ladder are shown in Supplemental Fig. S8D. Reprobing sequence for the same membrane is indicated with blue arrow.